RESEARCH Open Access

Neurotrophic support by traumatizedmuscle-derived multipotent progenitorcells: Role of endothelial cells and VascularEndothelial Growth Factor-AHeidi R. H. Zupanc1,2, Peter G. Alexander2 and Rocky S. Tuan1,2*

Abstract

Background: Adult mesenchymal stem cells (MSCs) have been shown to increase nerve regeneration in animal modelsof nerve injury. Traumatized muscle-derived multipotent progenitor cells (MPCs) share important characteristics with MSCsand are isolated from severely damaged muscle tissue following surgical debridement. Previous investigations haveshown that MPCs may be induced to increase production of several neurotrophic factors, suggesting thepossible utility of autologous MPCs in peripheral nerve regeneration following injury. Recent findings havealso shown that components of the vascular niche, including endothelial cells (ECs) and vascular endothelialgrowth factor (VEGF)-A, regulate neural progenitor cells and sensory neurons.

Methods: In this study, we have investigated the neuroinductive activities of MPCs, particularly MPC-producedVEGF-A, in the context of an aligned, neuroconductive nerve guide conduit and the endothelial componentof the vascular system. Embryonic dorsal root ganglia (DRG) seeded on poly-ϵ-caprolactone aligned nanofibrous scaffold(NF) constructs and on tissue culture plastic, were cocultured with induced MPCs or treated with theirconditioned medium (MPC-CM).

Results: Increased neurite extension was observed on both NF and tissue culture plastic in the presenceof MPC-CM versus cell-free control CM. The addition of CM from ECs significantly increased the neurotrophic activityof induced MPC-CM, suggesting that MPC and EC neurotrophic activity may be synergistic. Distinctly higher VEGF-Aproduction was seen in MPCs following neurotrophic induction versus culture under normal growth conditions.Selective removal of VEGF-A from MPC-CM reduced the observed DRG neurite extension length, indicating VEGF-Ainvolvement in neurotrophic activity of the CM.

Conclusions: Taken together, these findings suggest the potential of MPCs to encourage nerve growth via aVEGF-A-dependent action, and the use of MPC-CM or a combination of MPC and CM from ECs for peripheralnerve repair in conjunction with NFs in a nerve guide conduit. Due to the ease of use, application of bioactive agentsderived from cultured cells to enhance neurotrophic support presents a promising line of research into peripheralnerve repair.

Keywords: Blast trauma, Muscle stem cells, Mesenchymal stem cells, Peripheral nerve repair, Neurotrophic factors,Paracrine mechanism, Endothelial cells, VEGF-A

* Correspondence: rst13@pitt.edu1Department of Bioengineering, Swanson School of Engineering, Universityof Pittsburgh, Pittsburgh, PA 15219, USA2Center for Cellular and Molecular Engineering, Department of OrthopaedicSurgery, University of Pittsburgh School of Medicine, 450 Technology Drive,Room 221, Pittsburgh, PA 15219, USA

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Zupanc et al. Stem Cell Research & Therapy (2017) 8:226 DOI 10.1186/s13287-017-0665-4

BackgroundWithin a critical time frame, nerve injury healing re-quires the careful orchestration of several independentbut related processes [1]. When a nerve injury surpassesthe length of a critical-sized defect, the ideal nerve heal-ing rate of about one inch per month [2] may be limitedby fibrotic scar formation. The target tissue slowly losesthe ability to recruit the recovering nerve or encouragereinnervation [3]. While several strategies have been de-veloped to stimulate bridging of the injury gap by accel-erating nerve growth, the clinical treatment of choice,grafting of autologous nerve or blood vessels, promisesno guarantee of recovery and requires precise and some-times unattainable donor tissue geometry as well as ataxing, second surgery for tissue harvest [4]. When au-tologous donor tissue is unattainable, patients can betreated with synthetic, biodegradable nerve guides as alast resort, but such guides are currently associated withless favorable outcomes [5].Exciting research in recent years has attempted to in-

crease the neurotrophic potential of these nerve guideconduits, the most sophisticated of which attempts tomimic the physical, chemical, and temporal aspects ofthe native repair processes. The most basic function ofthese guides isolates the damaged nerve, concentratingneurotrophic signals from the distal stump while elimin-ating unwanted, scar-forming immune cell infiltration[6]. This cellular isolation must also allow constant nu-trient exchange for the sensitive nerves. These compet-ing concerns have been addressed in recent years bytightly controlling the porosity and thickness of nerveguide conduits [6]. Within that context, substrate pat-terning or the incorporation of aligned fibers mimicbands of Büngner formed by proliferating, reparativeSchwann cells [7, 8]. Advancements in polymer chemis-try and refinements of techniques in fibrous scaffold fab-rication, such as electrospinning, allow the precisecontrol of the fiber diameter, alignment, and degradationrate [9, 10] with the ultimate goal of complete scaffoldreplacement by native tissue. The clinically approvedbiomaterial, poly-ε-caprolactone (PCL) is a biodegrad-able synthetic polymer that can be easily utilized for thefabrication of electrospun micro- or nanofibers [11],with a degradation rate in vivo comparable to the rate ofnative tissue replacement [12]. Beyond providing a per-missive architecture, PCL does little to increase nervegrowth. To address this relative inertness, stem cells orgrowth factor augmentations of these guides represent apopular topic of research, including the incorporation ofbioactive, neurotrophic factors normally secreted bySchwann cells [13]. These include nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), ciliaryneurotrophic factor (CNTF), and glial cell-derivedneurotrophic factor (GDNF). These factors can be

tethered to or encapsulated within the nerve guide con-duits for controlled release [8, 14, 15].The bioactivity of exogenously delivered factors may

degrade with time, and experiments have shown thatdirect transplantation of biologically responsive cellulargrowth factor sources, such as Schwann cells andSchwann cell-like bone marrow-derived mesenchymalstem cells (MSCs), can enhance nerve repair outcomes[16, 17]. It has been reported that nerve coculture withor treatment with conditioned medium (CM) derivedfrom Schwann cells, Schwann-like MSCs, or undifferen-tiated MSCs positively influenced the length and densityof neurons dissociated from chick embryonic dorsal rootganglia (DRG) [18, 19].Ancillary to supporting the damaged nerve, MSCs are

thought to signal the surrounding tissue and (possibly)invading immune cells with a resulting net positive effecton nerve defect healing [20, 21]. The use of allogeneicMSCs, while being tested for certain clinical therapies,faces intensifying scrutiny over their immunogenicity[22]. Current sourcing of sufficient numbers of autolo-gous MSCs also involves invasive secondary procedures.A safe and efficacious autologous MSC isolation method,integrated with standard surgical treatment methods, isthus highly desirable.Growing evidence suggests that skeletal muscle con-

tains multipotent stem or progenitor cells [23]. MDSCs(muscle-derived stem cells), a preplating selected celltype related to pericytes or mesenchymal stem cells,have been shown to possess multilineage differentiationabilities [24]. Recent studies have shown that carefulmuscle tissue and cell isolation from the viable marginsof surgical waste obtained during debridement of ablast-traumatized extremity yields a population ofmuscle-derived multipotent progenitor cells (MPCs)[25]. As described in this article, MPCs are defined bytheir isolation from traumatized muscle and rapid (24 h)adherence to tissue culture plastic. MPCs should not tobe confused with MDSPCs, which are derived from non-traumatized muscle and adhere slowly to collagen-coated surfaces [26]. In addition to other functions,MPC neurotrophic activities and neurotrophic factor ex-pression [27] in vivo are similar to those of widelyknown and clinically utilized bone marrow-derivedMSCs [28], suggesting that MPCs might be an ideal can-didate cell type for autologous nerve repair for victimswho have suffered severe trauma and thus present read-ily accessible traumatized tissue specimens. To enhancethe clinical utility of MPCs and the production of neuro-trophic factors, MPCs can be neurotrophically inducedfollowing culture in a defined medium [27]; CM ob-tained following this induction can then stimulate neur-ite outgrowth in embryonic DRG [29]. Recent work alsosuggests that MPCs might convey neurotrophic benefits

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through indirect means, as MPCs exhibit the ability tocontrol their local proteolytic microenvironment [30].One manner of MSC tissue support might be throughan association with vascular endothelial cells (ECs), bothin the maintenance of a stem cell niche and in endogen-ous regenerative processes [31–33].ECs are intimately connected with the development,

growth, and continued health of nerves in the neurovas-cular unit [17, 34]. Neural and vascular cells often utilizesimilar molecular pathways for physical guidance [35],which could be exploited by MSC- or MPC-basedtherapies. There is ample evidence that the EC ligands,vascular endothelial growth factor (VEGF)-A and angio-poietin (ANGPT)-1, also exert positive effects on neu-rons [36]. VEGF-A receptor (VEGFR)2 is expressed byneurons [37], and recent clinical studies have reportedthat VEGF-A promoted anatomical and functionalrecovery of injured peripheral nerves in the avascularcornea [38], but the role of VEGF-A signaling withrespect to MSC- and EC-associated peripheral nerverepair remains undefined.Extrapolating from these findings, we hypothesize that

MPCs positively modulate nerve growth activities andthat this effect could be augmented through interactionswith ECs and physical contact guidance. These interac-tions were investigated using the embryonic DRG modelin two experimental set-ups: (1) treatment of DRGs cul-tured on tissue culture plastic with one or more cell-conditioned media (CMs); and (2) coculturing of DRGsand MPCs seeded onto an aligned nanofibrous biomate-rial scaffold, simulating the microenvironment within apolymeric nerve conduit construct. The results reportedhere show that neurotrophic differentiation of MPCstrongly affected DRG neurite extension on nanofibers,regardless of the presence or absence of ECs. Resultsfrom the CM investigations revealed that VEGF-A orVEGF-A-associated molecules in MPC-CM, derivedfrom cultures under neurotrophic or growth conditions,were responsible for a large portion of the observedneurite length enhancement effect.

MethodsNanofiber construct formation and assessmentAligned nanofiber constructs (NFs) were deposited re-producibly on glass slides (Fisher, Pittsburgh, PA) byelectrospinning, at ~ 15 kV difference from a 22-Gneedle placed 15 cm from a rotating mandrel, an11.5% PCL (Sigma-Aldrich, St. Louis, MO) solutiondissolved in 1:1 tetrahydrofuran:dimethylformamide(Sigma-Aldrich) [39]. NFs were prepared for cell cul-ture by lining each edge with silicone sealant (RTV734; Dow, Midland, MI); following an overnight cure,residual solvent was removed by storage for an add-itional night under vacuum with dessicant. Each slide

was then disinfected with 70% ethanol (Fisher) and10 min of UV-V light exposure (100–280 nm) (LED-wholesalers; Hayward, CA), Finally they were rinsedwith Dulbecco’s phosphate-buffered saline (PBS; Invi-trogen, Carlsbad, CA). After preparation, NFs were in-cubated in PBS with either serial applications of 100 ng/ml poly-L-lysine and 10 μg/ml laminin (Sigma-Aldrich) at4 °C overnight, or with 10% fetal bovine serum (FBS; Invi-trogen) at 4 °C 2× overnight. Fiber thickness and orienta-tion were determined using scanning electron microscopyand plugins developed for ImageJ/Fij (BoneJ 1.4.0 [40];Directionality 2.0 [41]).

Culture of traumatized muscle-derived MPCs and ECsTraumatized muscle-derived MPCs, isolated as describedpreviously [28] (four male patients, average age 24 years),were expanded from low initial cell numbers to passage5–8 in tissue culture-treated flasks (Fisher, Nunc, Roches-ter, NY) in growth medium (GM): α-minimum essentialmedium (MEM) + 10% FBS + penicillin-streptomycin-fun-gizone (PSF) + 1 ng/ml basic fibroblast growth factor(FGF)-2 (all reagents from Invitrogen). Human dermalmicrovascular ECs (HUVECs) were obtained from theCenters for Disease Control and Prevention (via MaterialTransfer Agreement) [33] and cultured on tissue culture-treated flasks in EGM-2MV medium (Lonza, Basel,Switzerland). At 90% confluency, cells were trypsinized(Invitrogen) and passaged at 1 × 106 cells/T150 flask. Allcells were cultured at 37 °C under 5% CO2.Twenty-four hours after passage, CM was generated by

culturing cells (denoted GM-MPC or EC) for 48 h in basalmedium, containing Dulbecco’s modified Eagle’s medium(DMEM) + 1% insulin-transferrin-selenium-ethanolamine(ITS-X; Life Technologies, Carlsbad, CA) + penicillin-streptomycin (PS; Invitrogen). CM was denoted MPC-CM,EC-CM, or acellular control when derived from MPCs,ECs, or cell-free basal medium incubated with the samesubstrate, respectively. Following harvest, CM was preparedby centrifugation at 200 g, stored frozen at –80 °C untiluse, and thawed and centrifuged again just prior to use.

Neurotrophic inductionMPCs were seeded at 1 × 103 cells/cm2 on tissue cultureplastic or NFs and differentiated neurotrophically usinga modified 10-day protocol [27, 29, 42]. Twenty-fourhours after seeding, MPCs were incubated with GMsupplemented with 10 mM β-mercaptoethanol (Sigma)for 24 h, followed by 48 h in GM supplemented with10 mM β-mercaptoethanol + 35 ng/ml retinoic acid(Sigma). For the 6 following days, MPCs were incubatedwith neurotrophic medium (DMEM/Ham’s F12 + PSF(Invitrogen) supplemented with 2% FBS, 2% B-27 (Invi-trogen), 6 mg/ml retinoic acid, 1 ng/ml FGF-2 (Sigma),10 ng/ml platelet-derived growth factor (PDGF; Sigma),

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150 ng/ml heregulin (an isoform of neuregulin-1;Sigma), and 10 μM forskolin (Sigma)).Following neurotrophic differentiation, MPCs (desig-

nated nMPCs) were washed with PBS and either lysedwith TRiZol, or incubated with basal medium (seeabove) for 48 h to produce conditioned medium(nMPC-CM). For DRG coculture experiments, tissueculture plastic-cultured nMPCs were trypsinized andtransferred to DRG-containing fibers at a concentrationof 1 × 103 cells/cm2.

EC/MPC culture on nanofibrous constructsMPCs and ECs were seeded on FBS-coated NFs at vari-ous densities (cells/cm2) in cell-specific growth medium;medium was exchanged every 48 h. Cell viability wasassessed by quantifying the number of fluorescent live(green) and dead (red) cells visible within each micros-copy image after Live/Dead staining (Invitrogen). Fluor-escent cells were visualized with an Olympus invertedmicroscope equipped with a motorized stage controlledthrough MetaMorph. Whole cell numbers were deter-mined automatically for each image by setting thresholdsfor fluorescence intensity, cell size, and cell shape usingcustom macros written for ImageJ/Fiji [43]. Cell densitywas determined by scaling the number of live cells ob-served in each image to 1 cm2.

Nerve DRG culture and confirmation of MPC/ECneurotrophic activityDRGs were obtained via microdissection from incubationday 9 chicken embryos, and placed on poly-L-lysine- andlaminin-coated (Sigma) tissue culture plastic [44, 45].After 24 h in nerve growth medium (basic Eagle’smedium, 10% horse serum, 1 nM GlutaMax; Invitrogen)supplemented with NGF + epidermal growth factor (EGF)+ PDGF, DRGs were cultured for 4 additional days in acombination medium of 1:1 CM:DRG basal medium.CM-supplemented media were exchanged daily.To assess the effect of MPC or EC coculture on DRG

neurite extension, freshly isolated DRGs were seeded onsterile, aligned poly-L-lysine- and laminin-coated NFs.After 24 h in nerve growth medium, trypsinized MPCs,nMPCs, ECs, or a 1:1 xMPC-EC mixture were seededonto DRG-containing NFs at a final density of 1 × 104

cells/cm2 (1 × 105 per NF) in 1 ml of DRG basalmedium. Control cultures were incubated in only 1 mlof DRG basal medium. All media were exchanged dailyfor 4 additional days, and DRG neurite outgrowth wasassessed as described previously.

Selective VEGF-A depletionVEGF-A was selectively removed from thawed, centri-fuged, and filtered CM using neutralizing antibodies(mouse IgG, mouse aVEGF-A; R&D, Minneapolis, MN)

bound to SpinTrap Protein G beads (Sigma). Beads andbound antibodies/factors were removed from CM bycentrifugation at 200 g for 5 min. Bound VEGF-A waseluted from the beads, and selective binding was con-firmed via VEGF-A enzyme-linked immunosorbent assay(ELISA; R&D). VEGF-A-depleted or immunoglobulin(Ig)G-control CM were indicated with “+αVEGF-A” or“+IgG,” respectively.

Confirmation of neurotrophic inductionNeurotrophic factor production/secretion into CM wasassessed via sandwich ELISAs (R&D). RNA was isolatedwith TRiZol, reverse transcribed, and analyzed byreverse-transcription polymerase chain reaction (RT-PCR) for neural gene expression (Superscript III (Invi-trogen) 18S rRNA, BDNF, CNTF, GDNF, Nestin, NGF,VEGF-A (Qiagen, Hilden, Germany)).

Neurite outgrowth measurementTo assess the effect of CM or MPC/EC coculture on neur-ite outgrowth, DRGs were fixed and stained immunohis-tochemically for heavy neurofilament (NEFH; Sigma)(Secondary Alexa-Fluor antibodies; Invitrogen). DRGswere visualized with an Olympus inverted microscopeequipped with a motorized stage controlled throughMetaMorph. Resultant mosaic images were stitched usingGrid/Collection Stitching (Fiji) [46]. Each image repre-sented one replicate. The 10 longest NEFH-positive neur-ite extensions were measured from the geometric centerof the original DRG cluster (ImageJ).

Fig. 1 Neurite extension of dorsal root ganglia (DRGs) seeded ontissue culture plastic and cultured in the presence of conditionedmedium (CM) from multipotent progenitor cells (MPCs) and/orendothelial cells (ECs). An increase in DRG neurite extension wasdetected as a synergistic effect of the mixed (1:1) CM derived fromneurotrophically-induced MPCs (nMPC) and EC over the basal DRGmedia acellular control (Acell. control). n = 6; Tukey’s. Indicated pvalue applies to that condition versus all other conditions

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Statistical analysisAll data are presented as mean ± standard deviation, un-less noted. Statistical differences and p values were de-termined by one- or two-way analysis of variance(ANOVA) and Sidak’s or Tukey’s test, as appropriate.

ResultsNeurotrophic support by MPCs and ECs cultured ontissue culture plasticTo assess the relative trophic properties of the differentcell types, conditioned medium (CM) from the variouscell types or basal (control) medium was incubated withtissue culture plastic-seeded DRGs. In the presence ofCM derived from ECs or neurotrophically inducedMPCs, DRG neurite extensions increased slightly (butnot significantly) above control lengths (Fig. 1). By con-trast, DRG neurite extension lengths increased to al-most twice that of the basal medium control in thepresence of a combination of the CM from both celltypes. This finding suggested that a combination ofMPC and EC neurotrophic activities might better

support neurite extension on a nerve guide conduitthan either cell type in isolation.

Nanofiber conduit (NF) fabricationEfficient nanofiber-based physical guidance of neuriteoutgrowth requires the presence of appropriately sized(nanoscale) parallel fibers. Because batch-to-batchconsistency of electrospun fibers is notoriously low [47],randomly selected scaffolds from multiple batches ofNFs were examined using scanning electron microscopy.Fiber diameter was quantified through image analysisand suggested fairly consistent nanofiber diameter (580± 280 nm) and relatively good alignment (22 ± 17o

dispersion).

Cell viability of effector cells on NFsTo ensure that DRG-effector cell cocultures includedsufficient space and nutrients for all cell types, includingoxidative stress-sensitive nerve cultures [48, 49], effectorcells were seeded on NFs and assessed for their long-term (>24 h) viability and density (cells/cm2). MPCs or

Fig. 2 Viability and density of multipotent progenitor cells (MPCs) seeded on aligned NF scaffold. Edge-to-edge mosaic images of NF-seeded MPCsstained with Live/Dead (green/red) assay were obtained at 24 h post-seeding at (a,e,i) 0.5, (b,f,j) 1, (c,g,k) 5, and (d,h,l) 10 × 103 (k) cells/cm2; scale bar= 100 μm. Evidence of NF-mediated cell alignment could be seen in the common orientation of the MPCs. e–h MPC density (presented as an exponentialscale) as a function of culture time. MPC density scaled with initial seeding density at early time points (days 1 and 3), but eventually converged towards1 k cells/cm2 by day 9, except for the 10 k cultures. Cell aggregation on the scaffolds resulted in extreme local variations in cell density. MPC viability (i–l)varied widely at early time points. By day 9, 65% or more cells remained viable, and viability increased with initial seeding density

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ECs were initially seeded on 10 cm2 serum-coated NFsat varying densities (0.5, 1, 5, 10 × 103 cells/cm2). Livecell density and percent viability were assessed dailyusing metabolic stain (Live-Dead stain) for the first3 days and after an additional week in culture (Fig. 2),corresponding to the schedule of neurotrophic induc-tion. Cells were cultured in their respective growthmedia to allow for maximum proliferation.MPCs tolerated NF culture well, reaching an equilib-

rium density of approximately 1–5 × 103 cells/cm2, butEC coverage and viability dropped steadily over theculture period. Cell aggregation contributed to large var-iations in within- and between-sample viability anddensity measurements.Because of these findings, subsequent experiments in-

volving NF scaffolds were examined after short culture(< 5 day) and at low-moderate initial cell densities (1 ×103/cm2). To examine longer term (> 5 days) effects onneurite extension and neurotrophic differentiation, ECsand MPCs were cultured on tissue culture plastic.

Influence of effector cell coculture on DRG neuriteextensionTo assess the potential neurotrophic effect of the com-bination of MPCs/nMPCs and ECs (effector cells) in thecontext of a nerve guide, DRGs and effector cells were

seeded onto NFs and cocultured. Prepared NFs werefirst seeded with DRGs. Twenty-four hours after DRGseeding on the NFs, cocultures were constructed byadding effector cells. Effector cells consisted of ECs,noninduced MPCs, nMPCs, and 1:1 EC-xMPC mix(co-effectors). Effector cells were added to the NFs at afinal effector cell density of 1 × 103 cells/cm2. NEFH-positive DRG neurite extensions were assessed after 4 daysof coculture with daily medium changes (Fig. 3).In the presence of MPC effector cells and regardless of

the MPC neurotrophic induction, DRG neurite exten-sions increased. Specifically, DRG neurite lengths innMPC-DRG cocultures were ~ 3- to 4-fold longer thanDRG neurite lengths observed in control cultures (NF-seeded DRGs with no effector cells). This finding confirmedthe functional utility of the neurotrophic differentiationprotocol. The additional presence of ECs on the NFs causeda trend towards an increase in DRG neurite length with thelongest extensions observed under co-effector cocultureconditions. This effect suggests that a nerve guide conduitcould promote nerve growth by incorporating a combin-ation of MPCs and ECs.

NF effects on effector cell neurotrophic activitiesTo assess whether NF seeding might have altered MPCneurotrophic functions, MPCs were differentiated on NF

Fig. 3 Neurite extensions of dorsal root ganglia (DRGs) seeded on nanofiber NF scaffold upon coculture with multipotent progenitor cells(MPCs) or combination of MPCs/endothelial cells (ECs) (1:1). The presence of MPCs, particularly after their neurotrophic induction, increased the lengthof DRG neurite extensions (a) over DRG control (acellular (Acell.) control/basal medium) cultures (b). EC coculture (c) slightly but not significantlyincreased DRG neurite extension length. MPC and neurotrophically-induced MPC (nMPC) coculture significantly increased neurite extension lengthwhen compared to the control. For xMPC-DRG coculture, EC coculture slightly but insignificantly increased neurite extension length. DRGs coculturedwith both nMPC and EC (nMPC-EC-DRG cocultures; d) exhibited significantly longer neurite extensions than MPC-DRG or MPC-EC-DRG cocultures.a n = 4; Sidak’s p values as indicated. b–d Scale bars= 400 μm for representative NEFH-stained images

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and tissue culture plastic by seeding cells in inductionmedium onto each surface at densities of 1 × 103 cells/cm2.Except for an increase in VEGF-A expression, neuro-trophic gene expression of NF-seeded nMPCs didnot change significantly compared to tissue cultureplastic-seeded nMPCs (Fig. 4a and b).However, pooled CM from NF-seeded nMPCs exhib-

ited much lower factor concentrations than CM fromsimilarly induced tissue culture plastic-seeded nMPCs.CM derived from tissue culture plastic-cultured nMPCscontained FGF-2 (130 ± 220 pg/ml, n = 6) and GDNF(30 ± 50 pg/ml) inconsistently, with multiple samplesyielding undetectable amounts of each factor. Similar toBDNF, FGF-2 and GDNF could not be detected in CMderived from NF-cultured nMPCs, and secreted CNTFand NGF could not be detected in any samples.When compared to culture on tissue culture plastic,

EC culture on NF resulted in significant changes in ECneurotrophic gene expression (Fig. 4c and d). Despitethese gene expression changes, only BDNF and VEGF-Acould be detected in EC-CM from either substrate (tis-sue culture plastic or NF). NF culture severely reducedthe level of neurotrophic growth factors measured inEC-CM. This observation and the above note of dimin-ished cytokine and growth factor concentrations in NF-seeded cultures suggests that protein-scaffold interac-tions may restrict cytokine and growth factor release[50]. This likely explains the diminished relative neuriteextension observed upon MPC or EC coculture with

DRGs (Fig. 3) versus tissue culture plastic-derived CMneurotrophic support (Fig. 1).

Mechanism of action of ECs and MPCsInterestingly, our analysis showed that VEGF-A, mostcommonly known for its role in angiogenesis and knownto be expressed robustly by MPCs, was secreted byMPCs in copious amounts upon neurotrophic induction(Fig. 4b and d). The magnitude of secreted VEGF-A rela-tive to other neurotrophic factors as well as the observa-tion of VEGFR [51] on the surface of nerves suggestedthat it might play a more significant role in MPC-mediated neurotrophic activity than previously realized.To test whether VEGF-A was acting as a mediator ofthe MPC effect on DRG, VEGF-A was selectively de-pleted from nMPC-CM, EC-CM, and VEGF-A-spikedmedium using anti-VEGF-A antibodies bound to ProteinG beads. Effective VEGF-A immunodepletion was con-firmed by ELISA (Fig. 5i). VEGF-A concentrations in con-trol and IgG-treated medium were identical (~ 170 pg/ml),and VEGF-A concentrations in VEGF-A-depleted sampleswere below the detection limit. VEGF-A removal fromCM derived from nMPCs (Fig. 5d) and ECs (Fig. 5f)significantly decreased the maximum observed length ofDRG neurite extensions when cultured on tissue cultureplastic (Fig. 5a–g), suggesting a dependence of the neuro-trophic activity of both cell types on VEGF-A in additionto other MPC-secreted neurotrophic compounds.

Fig. 4 Expression and production of neurotrophically induced MPCs (nMPC) and endothelial cells (EC) cultured on tissue culture plastic (TCP) andnanofiber constructs (NF). a,c NF-cultured growth factor and neurotrophic marker gene expression, assayed by RT-PCR and normalized to TCPcultures: secreted brain-derived neurotrophic factor (BDNF) (b) or vascular endothelial growth factor (VEGF) (d) levels from cells seeded on TCP orNF as measured by ELISA. nMPC (a) and EC (c) neurotrophic gene expression was mildly or significantly increased by NF culture in almost allcases. CM from cultures on NF contained lower levels of secreted growth factors than conditioned medium (CM) from TCP-cultured cells (b,d).Noninduced multipotent progenitor cells (MPCs) secreted more VEGF than nMPCs (b,d). n = 6; Student’s t test *p < 0.05, **p < 0.01, versus controls(a,c) or indicated groups (b,d). CNTF ciliary neurotrophic factor, GNDF glial cell-derived neurotrophic factor, NGF nerve growth factor

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DiscussionWe have previously reported that neurotrophic inductionof MPCs enhances their production of neurotrophic fac-tors, including BDNF and CNTF. Extending that work, re-sults from this study show that nMPCs could supportincreased DRG neurite extension on nanofiber conduitswith approved nerve guide materials. This support waspartially mediated through soluble factors, including thosefactors found in conditioned medium and secreted duringMPC-DRG coculture. This support was observed regard-less of the substrate on which the DRGs were seeded (tis-sue culture plastic or nanofiber scaffolds (NFs)), despiteadverse effects of the NFs on MPC viability and measur-able growth factors in the CM. Due to high between-patient and between-experiment variability, consistent andsignificant differences in VEGF-A or BDNF concentra-tions in the CM were not observed. When compared toMPCs, nMPCs were associated with distinctly higherVEGF-A concentrations as well as consistent BDNF pro-duction, similar to results reported in other work [16].

Our findings show that the combination of nMPCsand ECs preferentially encourages neurite elongation(Fig. 1). Signaling molecules from each cell type work al-most synergistically despite similar levels of knownneurotrophic factors, BDNF and VEGF-A, in the re-spective CMs (Fig. 4b and d). This enhanced neuro-trophic activity could be explained in part by a mixedpopulation of cells in the isolated MPCs. A CD29+/CD34+ vascular endothelial progenitor cell (EPC)-likepopulation has been noted within muscle isolates [52];muscle-derived stem cells have additionally been ob-served to spontaneously express both vascular andneurotrophic markers upon culture with neurons [53],suggesting plasticity, cell fusion, or a culture of mixedcell types [53, 54].For this investigation, nMPCs were investigated as local

sources of neurotrophic factors in the context of an im-planted NF. In keeping with previous studies, nMPC-CMwas utilized without concentration [29]. Other researchershave reported greatly enhanced MSC trophic activities

Fig. 5 Vascular endothelial growth factor (VEGF)-A production by neurotrophically induced MPCs (nMPC) and endothelial cells (EC) positively affectsneurite extension. a,c,e Dorsal root ganglia (DRG) neurite extensions in the presence of conditioned medium (CM) and immunoglobulin G (IgG) controlantibody. b,d,f DRG neurite extensions in the presence of CM and α-VEGF-A antibodies (VEGF-A immunodepletion). Compared to nonspecific IgGcontrol (a), selective removal of VEGF-A from a positive control consisting of exogenous VEGF-A (b) resulted in the loss of neurite growth induction,validating the effectiveness of VEGF-A immunodepletion. VEGF-A immunodepletion of CM derived from nMPC (d) and EC (f) decreased observedneurite extensions compared to IgG-treated mixtures (c and e, respectively), indicating that VEGF-A is an active component that contributes to bothMPC and EC neurotrophic activities. h Quantification of neurite growth following depletion of VEGF-A. VEGF-A immunodepletion was associated witha significant decrease in CM-induced neurite outgrowth (one-way ANOVA, Tukey’s). i VEGF-A levels in CM measured by ELISA. VEGF-A was present inCM derived from both MPCs and ECs and could be removed by immunoprecipitation using VEGF-A-specific antibodies (versus control IgG antibodies).a–g Representative NEFH-stained images; scale bar = 1 mm. h,i n = 3; one-way ANOVA Tukey’s *p < 0.05, **p < 0.01

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upon culture with cellulose fiber-concentrated CM [55],and it is possible that concentrating CM in future studiesmay yield stronger neural support.To promote better DRG attachment and growth on

NF substrate, laminin and lysine coatings were utilizedin line with a growing consensus that many aligned fi-brous structures bearing extracellular matrix (ECM)molecules are beneficial to the ultimate function of anerve guide conduit [54]. Several groups have also inves-tigated physical modifications to the PCL surfaces orchemical/ligand conjugation [12, 56] to better supportinitial nerve attachment and proliferation, and work is on-going with coatings derived from various ECM moleculesto increase MPC scaffold-based neurotrophic activity.Despite PCL NF culture of MPCs being unfavorable withrespect to neurotrophic induction (Fig. 2), construct-seeded MPCs both survived on the NFs (Fig. 2) and en-couraged increases in DRG neurite extension. Further-more, the combination coculture of ECs and nMPCsencouraged the longest neurite extensions (Fig. 3). In re-cent work with adipose-derived MSCs implanted in rats,Kingham et al. showed that the neurotrophic differenti-ation of those cells simultaneously promoted both EC vas-cular and DRG nerve function [57]. Taken together, thesefindings suggest that by optimizing the interactions ofMPCs and ECs preferential neural tissue growth could beachieved. Proteomic analysis of the secretomes of both celltypes might elucidate synergistic neurotrophic interactionsor potential antagonistic interactions between inhibitoryfactors in the CM of either the MPCs or ECs, the detailsof which warrant further study.Neurotrophic and angiogenic factors have been found

at widely varying levels within MSC- and muscle-derivedstem cell secretomes, depending on the donor and tissuesource [58, 59]. We therefore examined the potential in-volvement of VEGF-A in the neurotrophic potential ofCM from ECs and nMPCs. Following immunodepletionof VEGF-A from CM, neurite extension length de-creased when compared to the use of control antibodies,suggesting that VEGF-A is required for EC and nMPCpromotion of neurite outgrowth (Fig. 5). This finding isconsistent with other reports on the positive effect ofVEGF-A on DRG neurite extension, and this work re-ports the first analysis of potential mechanisms for MPCenhancement of neurite extension.

ConclusionTaken together, these findings demonstrate that theneurotrophic activities of MPCs are enhanced by bio-logical induction in vitro, CM combination with ECs, orcoculture with adult ECs. VEGF-A played a role in theobserved positive effect of MPC-CM on DRG neuriteextension. Given its dual role as an angiogenic andneurotrophic factor, coupled with the observation of

enhanced neurite extension under EC influence, VEGF-A should be incorporated in future NF-based cultures inan attempt to support both cell types. The unfavorableeffect of the fibrous scaffold on MPC gene expressionand measured protein secretion indicates that PCL,without appropriate ECM coating, is likely a suboptimalsubstrate for nMPCs. Testing of more cell-friendly sub-strates as well as coating the existing PCL scaffold struc-ture with various ECM molecules are both ongoing andshould yield useful information for the development of aregenerative construct for nerve repair.

AbbreviationsBDNF: Brain-derived neurotrophic factor; CM: Conditioned medium;CNTF: Ciliary neurotrophic factor; DMEM: Dulbecco’s modified Eagle’smedium; DRG: Dorsal root ganglion; EC: Endothelial cell; ECM: Extracellularmatrix; EGF: Epidermal growth factor; ELISA: Enzyme-linked immunosorbentassay; FBS: Fetal bovine serum; FGF: Fibroblast growth factor; GDNF: Glialcell-derived neurotrophic factor; GM: Growth medium; Ig: Immunoglobulin;MPC: Multipotent progenitor cell; MSC: Mesenchymal stem cell; NEFH: Heavyneurofilament; NF: Nanofiber scaffold; NGF: Nerve growth factor;nMPC: Neurotrophically induced multipotent progenitor cell; PBS: Phosphate-buffered saline; PCL: Poly-ε-caprolactone; PDGF: Platelet-derived growthfactor; PSF: Penicillin-streptomycin-fungizone; VEGF: Vascular endothelialgrowth factor; VEGFR: Vascular endothelial growth factor receptor;xMPC: MPC or nMPC

AcknowledgementsThis work would not have been possible without the help, advice, support,and/or guidance of the following individuals (listed alphabetically): NatashaBaker, Allison Bean, Donna Beer Stolz, Rachel Brick, Karen L. Clark, ThaisCuperman-Pohl, Solvig Diederichs, Jonathan Franks, Riccardo Gottardi,Thomas P. Lozito, Jian Tan, and Simon Watkins.

FundingFunding support is derived in part from the Commonwealth of PennsylvaniaDepartment of Health and US Department of Defense (W81XWH-10-2-0084,W81XWH-15-1-0600). HRHZ was a predoctoral trainee on a NIH T32 EB001026(CATER) Training Grant.

Availability of data and materialsThe datasets used and/or analyzed during the current study are availablefrom the corresponding author upon reasonable request.

Authors’ contributionsRST and PGA obtained the funding for this work, designed a portion of theexperiment, advised about data analysis and reporting, and edited themanuscript for content and clarity. HRHZ designed a portion of theexperiments, performed the experiments, analyzed and reported thedata, wrote the manuscript, and edited the manuscript for content andclarity. A portion of this work was presented in the Ph.D. dissertation ofHRHZ (as HRH). All authors read and approved the final manuscript.

Ethics approval and consent to participateMPCs were derived from debrided muscle waste during surgery on blast-traumatized individuals, with patient consent, at the Walter Reed ArmyMedical Center, according to an IRB-approved protocol (06-24046(2)), andused in this study with approved inter-institutional Material Transfer Agreementand according to an IRB approved, exempted protocol (PRO10090075). AlthoughIACUC approval is not required for the use of avian embryos, research with DRGsfrom avian embryos was performed in a manner that minimizes the suffering ordiscomfort of the avian embryo.

Consent for publicationNot applicable.

Zupanc et al. Stem Cell Research & Therapy (2017) 8:226 Page 9 of 11

Competing interestsRST is an Editor-in-Chief of the journal. The authors declare that they haveno competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in publishedmaps and institutional affiliations.

Received: 27 April 2017 Revised: 4 September 2017Accepted: 8 September 2017

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